17-371 EZ-ChIP™

17-371
22 assays  Kit capacity: 22 chromatin immunoprecipitation assays
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      For target-specific spike-in controls that make ChIP experiments more quantitative and accurate, Click on the Related Product & Applications tab above.
      Description
      Catalogue Number17-371
      Trade Name
      • EZ-ChIP
      DescriptionEZ-ChIP™
      OverviewChromatin Immunoprecipitation (ChIP) is an important technique allowing the researcher to analyze in vivo interactions of proteins with genomic DNA. Any chromatin-associated or DNA binding protein can be analyzed with this technique, provided a good antibody to the protein exists. One can measure different proteins localized to a specific region of the genome, or the genome wide distribution of a specific protein. Another powerful application of this technique is to analyze changes in histone modifications that correlate with processes like transcription, mitosis or DNA repair.

      Features & Benefits:
      Easier: Spin columns make DNA purification easier and more reliable - no more messy phenol-chloroform extractions.
      Quicker: All reagents to process your samples are included - you don't have to spend valuable time making them.
      Greater Reproducibility: Positive and negative control antibodies and PCR primers are included to help validate your results and to troubleshoot your experiments.
      Alternate Names
      • Agarose ChIP Kit
      Background InformationChromatin Immunoprecipitation (ChIP) is a powerful technique for mapping the in vivo distribution of proteins associated with chromosomal DNA. These proteins can be histone subunits and post-translational modifications or other chromatin associated proteins such as transcription factors, chromatin regulators, etc. Additionally, ChIP can be used to identify regions of the genome associated with these proteins, or conversely, to identify proteins associated with a particular region of the genome. ChIP methodology often involves protein-DNA and protein-protein cross-linking, fragmentation of the cross-linked chromatin, and subsequent immunoprecipitation of chromatin with an antibody specific to a target protein. The DNA fragments isolated in complex with the target protein can be identified by a variety of methods including PCR, DNA microarray and DNA sequencing. Standard or quantitative PCR can be performed to verify whether a particular DNA sequence (the gene or region of the genome) is associated with the protein of interest. The combination of ChIP and promoter or genomic tiling microarrays (ChIP-chip) allows genome-wide identification of DNA-binding sites for chromatin-associated proteins with precise resolution. Alternatively, high-throughput sequencing of libraries constructed from immunoprecipitated chromosomal DNA (ChIP-Seq) is a powerful alternative to ChIP-chip in mapping the protein-DNA interactions across mammalian genomes.
      References
      Product Information
      Components
      • DNA Purification Spin Columns and Collection Tubes
      • ChIP Blocked Protein G Agarose
      • Anti-RNA Polymerase II
      • Control PCR Primers
      • Normal Mouse IgG
      • Bind, Wash and Elution Reagents
      • Protease Inhibitor Cocktail II
      • RNase A
      • Proteinase K
      • All required buffers
      • ChIP Dilution Buffer
      • Low Salt Immune Complex Wash Buffer
      • High Salt Immune Complex Wash Buffer
      • LiCl Immune Complex Wash Buffer
      • TE Buffer
      • 0.5M EDTA
      • 5M NaCl
      • SDS Lysis Buffer
      • 1M Tris-HCl, pH 6.5
      • 10X PBS
      • 10X Glycine
      • 1M NaHCO3
      • Control Primers
      • 20% SDS
      • Spin Filters
      • Collection Tubes
      • Bind Reagent A
      • Wash Reagent B
      • Elution Reagent C
      PresentationContains all necessary reagents to perform 22 individual chromatin immunoprecipitation (ChIP) reactions. Supplied buffers are sufficient to generate chromatin from up to five 15 cm plates of cultured cells, each plate providing up to 10 chromatin preparations (varies with cell and assay type).
      Applications
      ApplicationThis EZChIP kit contains all necessary reagents to perform 22 individual chromatin immunoprecipitation (ChIP) reactions using inexpensive protein G agarose beads. Control primers included.
      Biological Information
      Analytes Available
      • Protein G
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Usage Statement
      • Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
      Storage and Shipping Information
      Storage ConditionsUpon receipt, store components at the temperatures indicated on the labels. Kit components are stable for 1 year from date of shipment when stored as directed.
      Packaging Information
      Material Size22 assays
      Material PackageKit capacity: 22 chromatin immunoprecipitation assays
      Transport Information
      Supplemental Information
      Specifications

      Documentation

      EZ-ChIP™ MSDS

      타이틀

      물질안전보건자료(MSDS) 

      EZ-ChIP™ Certificates of Analysis

      TitleLot Number
      EZ ChIP Chromatin Immunoprecipitation Kit - 21357012135701
      EZ ChIP Chromatin Immunoprecipitation Kit - 19593031959303
      EZ ChIP Chromatin Immunoprecipitation Kit - 19826521982652
      EZ ChIP Chromatin Immunoprecipitation Kit - 19938741993874
      EZ ChIP Chromatin Immunoprecipitation Kit - 19996211999621
      EZ ChIP Chromatin Immunoprecipitation Kit - 20033642003364
      EZ ChIP Chromatin Immunoprecipitation Kit - 20145302014530
      EZ ChIP Chromatin Immunoprecipitation Kit - 20251322025132
      EZ ChIP Chromatin Immunoprecipitation Kit - 20296792029679
      EZ ChIP Chromatin Immunoprecipitation Kit - 20328482032848

      References

      Reference overviewApplicationSpeciesPub Med ID
      FOXQ1 controls the induced differentiation of melanocytic cells.
      Bagati, A; Bianchi-Smiraglia, A; Moparthy, S; Kolesnikova, K; Fink, EE; Kolesnikova, M; Roll, MV; Jowdy, P; Wolff, DW; Polechetti, A; Yun, DH; Lipchick, BC; Paul, LM; Wrazen, B; Moparthy, K; Mudambi, S; Morozevich, GE; Georgieva, SG; Wang, J; Shafirstein, G; Liu, S; Kandel, ES; Berman, AE; Box, NF; Paragh, G; Nikiforov, MA
      Cell Death Differ  25  1040-1049  2018

      요약 표시
      29463842 29463842
      DeSUMOylation switches Kaiso from activator to repressor upon hyperosmotic stress.
      Zhenilo, S; Deyev, I; Litvinova, E; Zhigalova, N; Kaplun, D; Sokolov, A; Mazur, A; Prokhortchouk, E
      Cell Death Differ  0  2018

      요약 표시
      29472715 29472715
      Alcohol dysregulates miR-148a in hepatocytes through FoxO1, facilitating pyroptosis via TXNIP overexpression.
      Heo, MJ; Kim, TH; You, JS; Blaya, D; Sancho-Bru, P; Kim, SG
      Gut  0  2018

      요약 표시
      29475852 29475852
      Inhibition of MicroRNA-23b Attenuates Immunosuppression in Late Sepsis through NIK, TRAF1 and XIAP.
      Zhang, H; Li, H; Shaikh, A; Caudle, Y; Yao, B; Yin, D
      J Infect Dis  0  2018

      요약 표시
      29506272 29506272
      Precise nanoinjection delivery of plasmid DNA into a single fibroblast for direct conversion of astrocyte.
      Park, HS; Kwon, H; Yu, J; Bae, Y; Park, JY; Choi, KA; Choi, Y; Hong, S
      Artif Cells Nanomed Biotechnol  0  1-9  2018

      요약 표시
      29506416 29506416
      HPSE enhancer RNA promotes cancer progression through driving chromatin looping and regulating hnRNPU/p300/EGR1/HPSE axis.
      Jiao, W; Chen, Y; Song, H; Li, D; Mei, H; Yang, F; Fang, E; Wang, X; Huang, K; Zheng, L; Tong, Q
      Oncogene  37  2728-2745  2018

      요약 표시
      29511351 29511351
      Long noncoding RNA CCAT2 is activated by E2F1 and exerts oncogenic properties by interacting with PTTG1 in pituitary adenomas.
      Fu, D; Zhang, Y; Cui, H
      Am J Cancer Res  8  245-255  2018

      요약 표시
      29511595 29511595
      DJ-1 is involved in epigenetic control of sphingosine-1-phosphate receptor expression in vascular neointima formation.
      Lee, KP; Baek, S; Jung, SH; Cui, L; Lee, D; Lee, DY; Choi, WS; Chung, HW; Lee, BH; Kim, B; Won, KJ
      Pflugers Arch  0  2018

      요약 표시
      29511860 29511860
      miR-25-3p, Positively Regulated by Transcription Factor AP-2α, Regulates the Metabolism of C2C12 Cells by Targeting Akt1.
      Zhang, F; Chen, K; Tao, H; Kang, T; Xiong, Q; Zeng, Q; Liu, Y; Jiang, S; Chen, M
      Int J Mol Sci  19  2018

      요약 표시
      29518009 29518009
      GATA4 as a novel regulator involved in the development of the neural crest and craniofacial skeleton via Barx1.
      Guo, S; Zhang, Y; Zhou, T; Wang, D; Weng, Y; Chen, Q; Ma, J; Li, YP; Wang, L
      Cell Death Differ  0  2018

      요약 표시
      29523871 29523871

      Brochure

      Title
      An Introduction to Antibodies and Their Applications
      Shaping Epigenetics Discovery - Epigenetics Product Selection Brochure

      Data Sheet

      Title
      Reprogramming Cell Fate and Function Novel Strategies for iPSC Generation, Characterization, and Differentiation

      FAQ

      QuestionAnswer
      How should I resuspend my pellet prior to PCR?You should resuspend your pellet in water and not TE as the EDTA found in the TE may interfere with PCR.
      How many PCR reactions can be done with this kit?There are enough primers and PCR buffer for 4 reactions per IP assuming a 20 microliter volume and assuming the primers are at the recommended concentration as stated in the manual.
      Is there ever a time when I do not need to cross-link Histones?In native ChIP, Histone H3 and Histone H4 do not need to be crosslinked as they are very tightly associated. Histone H2A and Histone H2B are not as tightly associated, but will still work in native ChIP.
      From where are the primer sequences derived for the kit?The primer sequences are based on the Human GAPDH promoter. The GenBank number is NT_009759.15, using nts:6497145-6498136.
      What were your conditions for PCR?Please see the manual for The EZ ChIP Kit (Catalog #17-371) for more information.
      If I wanted to quantitate my immunoprecipitated DNA, how would I do so?DNA purified from ChIP experiments can be quantitated by PCR, providing the amplifying oligos meet specific criteria. Oligos should be 24 mers, with a GC content of 50% (+/- 4) and a Tm of 60.0C (+/- 2.0). You must be certain that the PCR reactions are within the linear range of amplification. Generally it takes time to achieve this. Too much input DNA will affect your results, so set up several tubes for each experiment to optimize the input DNA. Generally, this is about 1/25th to 1/100th for yeast, approximately 1/10 for mammalian cells, but depends on the amount of antibody and input chromatin.

      Also, do not use more than 20 cycles, making sure that dNTP's always remain in excess. Also, include each reaction a control primer (to compare your experimental band against-make sure the sizes are sufficiently different to allow proper separation-75 base pairs is usually OK) set to a region of the genome that should not change throughout your experimental conditions. Also PCR from purified input DNA (no ChIP) and include no antibody control PCR's as well. PCR products should be no more than 500 base pairs and should span the area of interest (where you think you will see changes in acetylation or methylation of histones). All PCR products should be run on 7-8% acrylamide gels and stained with SYBR Green 1 (Molecular Probes) at a dilution of 1:10,000 (in 1X Tris-borate-EDTA buffer, pH 7.5) for 30 minutes-no destaining is required.

      Quantitation is carried out subsequent to scanning of the gel on a Molecular Dynamics Storm 840 or 860 in Blue fluorescence mode with PMT voltage at 900 with ImageQuant software. This has distinct advantages over ethidium bromide staining. SYBR Green is much more sensitive, and illumination of ethidium stained gels can vary across the gel based on the quality of UV bulbs in your in your light box. For further info, see Strahl-Bolsinger et al. (1997) Genes Dev. 11: 83-93. A radioactive quantitation m
      I am not getting amplification with input DNA. What did I do wrong?Your input DNA sample should be taken just prior to adding the antibody. It is considered the starting material. If you are not seeing amplification with your input DNA, either you have not successfully reversed the cross links or the PCR is not working for reasons other than the kit.
      How would you recommend eluting Antibody-protein-DNA complexes from agarose (or sepharose) in order to perform a Re-ChIP experiment?The complex is removed with the elution buffer that you find in the ChIP assay kit. For a re-CHIP, it might make sense to add protease inhibitors to the IP wash buffers and the elution buffer and the second set of dilution buffers. Make sure everything stays cold so that the proteins aren't degraded during the collection of the first complex or during the second IP.
      Do you have any tips for sonication?Keep cells on ice throughout the procedure - even during sonication. Be sure that you don't sonicate for to long (more than 30 seconds could cause sample overheating and denaturation).
      Why is more DNA is precipitated in my no-antibody control than for my test sample?To eliminate banding in your negative controls you can do several things:

      A) Pre-clear the 2ml diluted cell pellet suspension with 80 microliters of Salmon Sperm DNA/Protein A Agarose-50% Slurry for 30 minutes at 4ºC with agitation. You could try to preclear the lysate longer or with more clearings.

      B) Titrate your input DNA, to see when the bands in the NFA disappear.

      C) Use an alternative lysis procedure: Resuspend cell pellet in 200 microliters of 5mM Pipes pH 8.0, 85mM KCl, 0.5% NP40 containing protease inhibitors. Place on ice for 10 minutes. Pellet by centrifugation (5 minutes at 5000 rpm). Resuspend pellet in 200 microliters of 1% SDS, 10mM EDTA, 50mM Tris-HCl, pH 8.1 containing protease inhibitors. Incubate on ice for 10 minutes.

      D) Block the Salmon Sperm DNA Agarose prior to use in 1-5% BSA and Chip dilution buffer (mix at room temperature for 30 minutes). After incubation, spin the agarose and remove the 1% BSA/ChIP assay buffer supernatant. Wash once in ChIP assay buffer and continue.

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      카테고리

      Life Science Research > Kits & Assays > ChIP Kits > Kits and Assays
      Life Science Research > Antibodies and Assays > Immunoassays > Immunoprecipitation (IP) > Chromatin Immunoprecipitation (ChIP) > ChIP Kits & Beads